Helicobacter therapeutic

ABSTRACT

The present invention relates to method of treating and/or preventing inflammation in a subject caused by bacteria that express HtrA. The present invention further relates to immunogenic compositions comprising an HtrA polypeptide or fragment thereof, as well as a therapeutic and/or prophylactic vaccine for treating or preventing disease.

FIELD

The present invention relates to a method of treating and/or preventinginflammation in a subject. The present invention further relates toimmunogenic compositions comprising a polypeptide or fragment thereof,as well as a therapeutic and/or prophylactic vaccine for treating orpreventing disease.

BACKGROUND

Helicobacter pylori are gram negative bacteria that are major pathogensof the human stomach. Infection typically occurs during childhood, withtransmission being most probably via the oral-oral route. Onceestablished, virtually all infections are for life and it is estimatedthat half the World's population is infected with these bacteria.

H. pylori infection drives a chronic gastritis that, in someindividuals, is the main aetiological factor in the development of arange of pathologies, including gastric ulcers, duodenal ulcers, gastricMALT lymphoma and gastric adenocarcinoma. Gastric adenocarcinoma remainsthe third leading cause of death worldwide due to malignancy, behindlung and liver cancer. It is fully accepted that the development ofthese H. pylori-associated diseases is a direct result of chronicgastritis and the more severe the inflammation the more likely it is forthese pathologies to develop.

H. pylori infection is currently treated by combination antibiotictherapy. However, the increasing development of antibiotic resistance isa serious concern. Failure rate for the first treatment is 20% and forthe second treatment is 25%, making an overall failure rate after 2rounds of treatment of 5%. With increasing rates of resistance, thisrate is predicted to increase in coming years.

Since its discovery, there has been a large effort to develop aneffective vaccine aimed at protecting against H. pylori infection. Theseefforts have thus far been completely unsuccessful. The main problempreventing the development of an effective vaccine is the inability toreliably produce sterilising immunity. There are a multitude of vaccineapproaches that will reduce H. pylori colonisation in animal models butnone that will completely eradicate it. Vaccinations of mice, eitherprophylactically or therapeutically, typically results in a 1-2 logreduction in bacterial numbers, while clinical trials have shown noevidence of any efficacy to date.

Moreover, protective immunity in mice induced by prophylacticvaccination is commonly associated with an increase in the severity ofgastritis (termed post-immunisation gastritis), although this has notbeen reported for therapeutic vaccination.

Accordingly, there remains a need for effective prophylactic andtherapeutic treatments of inflammation and diseases caused by H. pylori.

SUMMARY

The present inventors found that vaccinating a subject with an HtrApolypeptide reduces or prevents the development of bacteria inducedinflammation and disease despite persistence of the bacterial infectionpost vaccination.

Accordingly, a first aspect provides a method of treating or preventingbacteria induced inflammation in a subject, the method comprisingadministering to the subject a bacterial HtrA polypeptide or fragmentthereof of at least 10 amino acids, and/or an antibody that bindsbacterial HtrA.

In one embodiment of the first aspect, the method comprisesadministering an immunogenic composition comprising a bacterial HtrApolypeptide or fragment thereof of at least 10 amino acids.

A second aspect provides use of a bacterial HtrA polypeptide or afragment thereof of at least 10 amino acids, and/or an antibody thatbinds bacterial HtrA in the treatment or prevention of bacteria inducedinflammation in a subject.

A third aspect provides use of a bacterial HtrA polypeptide or afragment thereof of at least 10 amino acids, and/or an antibody thatbinds bacterial HtrA, in the manufacture of a medicament for thetreatment or prevention of bacteria induced inflammation in a subject.

In one embodiment of the first to third aspects, the inflammation isinduced by an enteropathogenic bacterium and the HtrA polypeptide is anenteropathogenic bacterial HtrA polypeptide or fragment thereof of atleast 10 amino acids. In one particular embodiment, the enteropathogenicbacterium is selected from Helicobacter pylori, Escherichia coli,Shigella flexneri, and Campylobacter jejuni and/or the enteropathogenicbacteria HtrA is selected from Helicobacter pylori, Escherichia coliShigella flexneri and Campylobacter jejuni HtrA.

In another embodiment of the first to third aspects, the inflammation isinduced by Helicobacter pylori and the HtrA polypeptide is aHelicobacter pylori HtrA polypeptide or fragment thereof of at least 10amino acids.

In another embodiment of the first to fourth aspects, the HtrApolypeptide or fragment thereof of at least 10 amino acids comprises anamino acid sequence at least 95% identical to any one of SEQ ID NOs:1-6or to a fragment thereof of at least 10 amino acids.

In another embodiment of the first to third aspects, the HtrApolypeptide or fragment thereof of at least 10 amino acids comprises anamino acid sequence at least 95% identical to any one of SEQ IDNos:7-36.

In another embodiment of the first to third aspects, the bacteriainduced inflammation is gastritis and/or duodenitis.

In yet another embodiment of the first to third aspects, theinflammation is atrophic gastritis.

A fourth aspect provides an immunogenic composition comprising abacterial HtrA polypeptide or a fragment thereof of at least 10 aminoacids, and/or an antibody that binds bacterial HtrA, for use in thetreatment or prevention of bacteria induced inflammation in a subject.

In one embodiment of the fourth aspect, the immunogenic compositioncomprises a bacterial HtrA polypeptide or a fragment thereof of at least10 amino acids.

In another embodiment of the fourth aspect, the bacterial HtrApolypeptide or fragment thereof of at least 10 amino acids is aHelicobacter pylori polypeptide or fragment of at least 10 amino acidsthereof.

In another embodiment of the fourth aspect, the composition comprises anHtrA polypeptide comprising an amino acid sequence at least 95%identical to any one of SEQ ID NOs:1-6 or a fragment thereof of at least10 amino acids.

In another embodiment of the fourth aspect, the HtrA polypeptide orfragment thereof of at least 10 amino acids comprises an amino acidsequence at least 95% identical to any one of SEQ ID Nos:7-36.

In another embodiment of the fourth aspect, the composition comprises apharmaceutically acceptable excipient and/or adjuvant.

In yet another embodiment of the fourth aspect, the immunogeniccomposition is a vaccine. In one particular embodiment, the immunogeniccomposition is a therapeutic vaccine.

A fifth aspect provides a pharmaceutical composition comprising anantibody that binds to bacterial HtrA polypeptide. Preferably, theantibody binds specifically to HtrA.

In one embodiment, the antibody binds Helicobacter pylori HtrApolypeptide.

In another embodiment of the fifth aspect, the antibody is a monoclonalantibody, a chimeric antibody, a humanised antibody, a human antibody,Fab, F(ab′)₂, or an scFv.

A sixth aspect provides use of the immunogenic composition describedherein, the pharmaceutical composition described herein, or an antibodythat binds bacterial HtrA in the manufacture of a medicament for thetreatment or prevention of bacteria induced inflammation.

In one embodiment of the sixth aspect, the bacteria induced inflammationis Helicobacter pylori induced inflammation.

In another embodiment of the sixth aspect, the bacteria inducedinflammation is gastritis and/or duodenitis. In one particularembodiment, the inflammation is atrophic gastritis.

A seventh aspect provides a method of making an immunogenic composition,the method comprising mixing an HtrA polypeptide or fragment thereof ofat least 10 amino acids with a pharmaceutically acceptable excipientand/or adjuvant.

In one embodiment of the seventh aspect, the method comprises isolatingan HtrA polypeptide. In one particular embodiment, the HtrA polypeptideis a recombinant HtrA polypeptide.

In an embodiment of the seventh aspect, the immunogenic composition is avaccine.

An eighth aspect provides a method of treating or preventing a diseasecaused by Helicobacter pylori infection in a subject, the methodcomprising administering to the subject an HtrA polypeptide or fragmentthereof of at least 10 amino acids.

In one embodiment of the eighth aspect, the method comprisesadministering to the subject an immunogenic and/or pharmaceuticalcomposition comprising an HtrA polypeptide or fragment thereof of atleast 10 amino acids.

In one embodiment of the seventh or eighth aspect, the HtrA polypeptideor fragment thereof of at least 10 amino acids is a Helicobacter pyloriHtrA polypeptide or fragment thereof of at least 10 amino acids.

In another embodiment of the seventh or eighth aspect, the HtrApolypeptide comprises an amino acid sequence at least 95% identical toany one of SEQ ID Nos:1-6 or a fragment thereof of at least 10 aminoacids.

In another embodiment of the seventh or eighth aspects, the HtrApolypeptide or fragment thereof comprises an amino acid sequence atleast 95% identical to any one of SEQ ID Nos:7-36.

In one embodiment of the eighth aspect, the immunogenic compositioncomprises a pharmaceutically acceptable excipient and/or adjuvant.

In another embodiment of the eighth aspect, the disease caused byHelicobacter pylori infection is inflammation, gastritis, gastric ulcer,duodenal ulcer, gastric cancer, and/or mucosa-associated-lymphoid-tissue(MALT) lymphoma.

In one embodiment of the first and eighth aspects, the method comprisesadministering an attenuated bacteria or virus comprising the HtrApolypeptide or fragment thereof of at least 10 amino acids to thesubject.

In one embodiment of the third to seventh aspects, the composition ormedicament comprises an attenuated bacteria or virus comprising the HtrApolypeptide or fragment thereof of at least 10 amino acids to thesubject.

A ninth aspect provides an attenuated bacterium or virus comprising anHtrA polypeptide or fragment thereof of at least 10 amino acids.

In one embodiment, the HtrA polypeptide or fragment thereof of at least10 amino acids is a Helicobacter pylori HtrA polypeptide or fragmentthereof of at least 10 amino acids.

In another embodiment, the HtrA polypeptide comprises an amino acidsequence at least 95% identical to any one of SEQ ID Nos:1-6 or afragment thereof of at least 10 amino acids.

In yet another embodiment, fragment thereof comprises an amino acidsequence at least 95% identical to any one of SEQ ID Nos:7-36.

A tenth aspect provides a DNA vaccine comprising a polynucleotideencoding a bacterial HtrA polypeptide or fragment thereof of at least 10amino acids.

In one embodiment, the polynucleotide encodes an HtrA polypeptide orfragment of at least 10 amino acids comprising an amino acid sequence atleast 95% identical to any one of SEQ ID NOs:1-36.

In another embodiment, upon administration of the DNA vaccine to asubject, the polypeptide is expressed and an immune response to thepolypeptide is produced.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Prophylactic vaccination with recombinant HtrA induces someprotection against H. pylori challenge. Groups of mice (n=6) werevaccinated twice via the nasal route with killed H. pylori plus choleratoxin (CT) adjuvant (positive control) or HtrA plus CT. Negativecontrols were sham dosed with PBS (n=5). Four weeks after the seconddose, all mice were challenged with live H. pylori strain SS1. Bacterialburdens in mouse stomachs were assessed four weeks later by colonyforming assay. Vaccinations reduced bacterial colonisation compared tothe negative control group (**p<0.01, ***p<0.001; ANOVA).

FIG. 2. Prophylactic vaccination with recombinant HtrA does not causepost-immunisation gastritis. Stomachs from the mice were assessedhistologically for gastritis. As expected, mice vaccinated with killedH. pylori plus CT before challenge had a significantly increasedseverity of gastritis compared to the negative control group(*Mann-Whitney). However the gastritis in mice vaccinated with HtrA plusCT was indistinguishable from the negative control group. In otherwords, despite the protective immunity shown in FIG. 1, these mice wereprotected from developing post-immunisation gastritis.

FIG. 3. Prophylactic vaccination with recombinant HtrA (rHtrA)suppresses gastric cytokine levels in response to H. pylori infection.Cytokine levels in the gastric tissues of the mice described in FIG. 1were quantified by ELISA. As expected, gastric tissues from theinfection control mice (PBS) and mice that received the positive controlvaccine (H. pylori+CT) presented with high cytokine levels includingthose known to be highly pro-inflammatory (IFNγ and IL-17A).Unexpectedly, vaccination with HtrA+CT significantly suppressed levelsof these cytokines in infected mice to extremely low or undetectablelevels (**p<0.01, ***p<0.001; ANOVA).

FIG. 4. Prophylactic vaccination with recombinant HtrA induces an enzymeneutralising antibody response. Sera were collected from thevaccinated/challenged mice shown in FIG. 1 at the end of the experiment(four weeks after H. pylori challenge). These sera were mixed with HtrAprior to measuring its proteolytic cleavage of casein. Sera from micevaccinated with HtrA then challenged with H. pylori inhibited theability of HtrA protease to cleave this protein substrate (***ANOVA), incontrast to sera from the other groups.

FIG. 5. Vaccination with HtrA induces serum antibodies against HtrA. Agroup of C57BL/6 mice (n=8) were vaccinated nasally with rHtrA plus CT.Three vaccinations were given, spaced by three weeks. A control groupwas sham-treated with PBS. One week after the last vaccination, micewere killed and sera collected. Antibodies against rHtrA was measured byELISA.

FIG. 6. Therapeutic vaccination with injected HtrA plus alum does notreduce colonization by H. pylori. Groups of C57BL/6 mice (n=7/8) wereinfected with H. pylori for four weeks before being subcutaneouslyinjected with four weekly doses of either alum adjuvant alone (negativecontrol), rHtrA and alum or rHtra^(sa) and alum. Four weeks after thelast vaccination, stomachs were removed and bacterial colonizationquantified by colony-forming assay.

FIGS. 7A-7B. Therapeutic vaccination with injected HtrA plus alumprotects against H. pylori induced gastritis. The severity of gastritisin stomachs of therapeutically vaccinated mice (as described in FIG. 6)were quantified histologically. A) rHtra and rHtrA^(sa) groups shownseparately. B) The two rHtrA groups combined. Percentages indicate theproportion of mice that had no visible cell infiltrate, metaplasia oratrophy. Despite having no effect on colonization (FIG. 6), vaccinationwith either active rHtra or inactive rHtrA^(sa) resulted in protectionagainst the development of H. pylori-induced atrophic gastritis.

FIG. 8. Therapeutic vaccination with injected HtrA plus alum protectsagainst H. pylori induced gastritis without a significant increase ingastric cytokines. Cytokine levels in the gastric homogenates from micetherapeutically vaccinated with HtrA (as described in FIG. 6) werequantified by ELISA. Vaccination with either rHtrA group produced nosignificant increase in key gastric cytokines, particularly comparedwith the main negative control group, which were vaccinated with alumalone.

FIG. 9. Therapeutic vaccination with injected HtrA plus alum inducesintestinal antibodies against native HtrA. Intestinal scrapings werecollected from mice described as in FIG. 6. Antibodies binding H. pyloriHtrA were detected by western blot. Blots are shown from negativecontrol mice (left uninfected and unvaccinated, or infected theninjected with alum alone) or H. pylori infected mice vaccinated withrHtrA and alum or rHtrA^(SA) and alum. Lanes show blots probed with serafrom individual mice.

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—Amino acid sequence of H. pylori HtrA polypeptide (Genbankaccession: DAA34967).

SEQ ID NO:2—Amino acid sequence of H. pylori HtrA polypeptide (Genbankaccession: EJC53274).

SEQ ID NO:3—Amino acid sequence of H. pylori HtrA polypeptide (Genbankaccession: EJC13564).

SEQ ID NO:4—Amino acid sequence of H. pylori HtrA polypeptide (Genbankaccession: EJC10337).

SEQ ID NO:5—Amino acid sequence of H. pylori HtrA polypeptide (Genbankaccession: EJC09766).

SEQ ID NO:6—Amino acid sequence of H. pylori HtrA polypeptide (Genbankaccession: EJC07480).

SEQ ID NOs:7-30—Amino acid sequences of H. pylori HtrA polypeptidefragments.

SEQ ID NO:31—Amino acid sequence of H. pylori HtrA polypeptide Trypsindomain.

SEQ ID NO:32-35—Amino acid sequences of H. pylori HtrA polypeptide PDZ 1domains.

SEQ ID NO:36—Amino acid sequence of H. pylori HtrA polypeptide PDZ 2domain.

SEQ ID NOs:37-40—Oligonucleotide primers

DETAILED DESCRIPTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in immunology,microbiology and biochemistry).

Unless otherwise indicated, the molecular genetics, biochemistry, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J,Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook and Russell., Molecular Cloning: A LaboratoryManual, 3^(rd) edn, Cold Spring Harbour Laboratory Press (2001), R.Scopes, Protein Purification—Principals and Practice, 3^(rd) edn,Springer (1994), T. A. Brown (editor), Essential Molecular Biology: APractical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover andB. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4,IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present), EdHarlow and David Lane (editors) Antibodies: A Laboratory Manual, ColdSpring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors)Current Protocols in Immunology, John Wiley & Sons (including allupdates until present).

The terms “treating”, “treat” or “treatment” as used herein includereference to administering an HtrA polypeptide or fragment thereof, anantibody that binds an HtrA polypeptide, or a composition or vaccine asdescribed herein to reduce or eliminate at least one symptom ofinflammation in a subject that is induced by a bacterium expressingHtrA.

The term “preventing” refers to protecting a subject that may be exposedor infected with bacteria expressing an HtrA polypeptide from developingat least one symptom of bacteria induced inflammation.

“Administering” as used herein is to be construed broadly and includesadministering a composition or therapeutic agent as described herein toa subject or patient as well as providing the composition or therapeuticagent to a cell, such as, for example, by the provision of a prodrug toa patient.

The terms “specifically binds”, “bind specifically”, “specific binding”refer to the ability of an antibody to bind to a target molecularspecies in preference to binding to other molecular species with whichthe specific binding agent and target molecular species are admixed.

HtrA Serine Protease

HtrA is a serine protease and periplasmic chaperone in a number ofenteropathogenic bacterial species including Helicobacter pylori,Escherichia coli, Shigella flexneri, and Campylobacter jejuni. Thus, theterms “HtrA polypeptide” or “HtrA polynucleotide” refer to a bacterialpolypeptide or polynucleotide that has been assigned as or identifiedas, or is predicted to be, an HtrA on a database of gene or proteinsequences such as Genbank or EMBL. In one embodiment, the HtrApolypeptide or polynucleotide is a sequence that belongs to the genomeof any one of Helicobacter pylori, Escherichia coli, Shigella flexneri,and Campylobacter jejuni on a sequence database. In a preferredembodiment, the HtrA polypeptide or polynucleotide is an H. pylori HtrApolypeptide or polynucleotide (i.e. a polypeptide or fragment thereof,or polynucleotide or fragment thereof, that is identified on a sequencedatabase as being an HtrA sequence in the genome of H. pylori).

To date, HtrA is the only identified serine protease produced by H.pylori. It is a ˜55 kDa protein that is both expressed on the bacterialsurface and secreted. It is essential to bacterial survival, even for invitro growth. Non-limiting examples of amino acid sequences of HtrApolypeptides include Genbank accession numbers DAA34967 (SEQ ID NO:1),EJC53274 (SEQ ID NO:2), EJC13564 (SEQ ID NO:3), EJC10337 (SEQ ID NO:4),EJC09766 (SEQ ID NO:5) and EJC07480 (SEQ ID NO:6).

The HtrA polypeptide used in the methods and compositions of theinvention may be an isolated polypeptide that is purified directly fromthe bacteria from which it is derived using standard techniques in theart.

In one embodiment, the polypeptide is a recombinant polypeptide. Theterm “recombinant” in the context of a polypeptide includes reference tothe polypeptide when produced by a cell in an altered amount or at analtered rate compared to its native state, as well as production of thepolypeptide in a cell that does not naturally produce the polypeptide.The term “recombinant” also refers to the production of a polypeptide ina cell-free expression system.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, (1970)) analysis (GCG program) with a gap creation penalty=5,and a gap extension penalty=0.3. The query sequence is at least 100amino acids in length, and the GAP analysis aligns the two sequencesover a region of at least 100 amino acids. In one example, the querysequence is at least 200 amino acids in length, and the GAP analysisaligns the two sequences over a region of at least 200 amino acids. Inone example, the GAP analysis aligns two sequences over their entirelength.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, more preferably at least 93%, more preferablyat least 94%, more preferably at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, morepreferably at least 99%, more preferably at least 99.1%, more preferablyat least 99.2%, more preferably at least 99.3%, more preferably at least99.4%, more preferably at least 99.5%, more preferably at least 99.6%,more preferably at least 99.7%, more preferably at least 99.8%, and evenmore preferably at least 99.9% identical to the relevant nominated SEQID NO.

Also included within the scope of the invention are immunogeniccompositions and/or vaccines comprising a fragment of an HtrApolypeptide. In one embodiment, the fragment is an “immunogenicfragment” of an HtrA polypeptide.

In an embodiment, the fragment of the HtrA polypeptide is at least 8amino acids in length, more preferably 9 amino acids in length, or morepreferably at least 10 amino acids in length. Alternatively, thefragment is at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50,60, 70, 80, 90 or 100 or more amino acids in length. Non-limitingexamples of fragments of HtrA polypeptides are provided as SEQ IDNos:7-36. Thus, in one embodiment, the HtrA polypeptide administered toa subject is able to elicit an immune response to a polypeptidecomprising an amino acid sequence selected from any one of SEQ IDNos:1-36.

As would be understood by the person skilled in the art, an immunogeniccomposition, pharmaceutical composition and/or vaccine may comprise adomain of the HtrA polypeptide, such as, for example, a Trypsin domain,PDZ 1 domain or PDZ 2 domain. Example amino acid sequences of HtrApolypeptide domains are provide as SEQ ID NO:31 (Trypsin 2 domain), SEQID Nos:32-35 (PDZ 1 domain) and SEQ ID NO:36 (PDZ 2 domain).

Immunogenic Compositions and Vaccines

Helicobacter pylori has been strongly linked to the development ofinflammation and gastric and duodenal ulcers, and it has been shown thateradication of H. pylori can prevent ulcers forming. Indeed patientspresenting with ulcers should be tested for H. pylori and treatedbecause eradication of H. pylori in patients with pre-existing ulcerscures ulcer disease and can prevent most recurrences. Gastricadenocarcinoma is the third leading cause of cancer death worldwide andthere is strong evidence that H. pylori contributes to the developmentof gastric cancer. Although other factors such as a diet low infruit/vegetables, smoking, age and a high salt intake also increase therisk of gastric cancer, H. pylori infection is most closely associatedwith stomach cancer. H. pylori infection can also lead to thedevelopment of a condition known as MALT Lymphoma a type of cancer ofthe stomach. Treatment and eradication of H. pylori infection can resultin regression of this latter malignancy in up to 75% of cases.

Conventional treatment for H. pylori infection is a 7-day course ofmedication called Triple Therapy comprising two antibiotics, amoxicillinand clarithromycin, to kill the bacteria together with an acidsuppressor to enhance the antibiotic activity. With the use ofantibiotics to treat so many patients with various conditions it hasbecome more difficult to treat H. pylori due to increasing occurrence ofantibiotic resistant strains. As a result, up to 25% of patients failthe first line therapy.

In view of the failures of conventional therapy for H. pylori infection,the present inventors have found that vaccination of a subject with anHtrA polypeptide or a fragment thereof reduces or prevents thedevelopment of inflammation and/or disease caused by the bacterium.Without wishing to be bound by any particular theory, it is hypothesizedthat vaccination against HtrA induces neutralizing antibodies that blockHtrA activity during infection. The lack of HtrA activity prevents theopening of the cell junctions and disruption of the epithelial barrier,thus preventing the transfer of bacterial components into the stomachtissue, protecting against induction of inflammation.

Accordingly, the present invention provides immunogenic compositions andvaccines for treating or preventing inflammation or a disease caused byinfection of a subject with a bacterium expressing an HtrA polypeptide.

An “immunogenic composition” as used herein refers to a composition thatcomprises an HtrA polypeptide or a fragment thereof and that elicits animmune response against the HtrA polypeptide or a fragment thereof. Theterm “immunogenic composition” also includes reference to a “vaccine”.The term “vaccine” covers any composition that induces an immuneresponse that is at least partially protective against inflammationand/or disease caused by the targeted pathogen or which efficaciouslyprotects against the pathogen and/or reduces colonization or infectionby the pathogen. By inducing an “at least partially protective” immuneresponse it is meant that a vaccine reduces or prevents bacteria inducedinflammation, infection and/or colonization by a bacterium expressing anHtrA polypeptide or reduces at least one symptom caused by infectionwith a bacterium expressing an HtrA polypeptide. An immunogeniccomposition may select, activate or expand cells of the immune systemincluding memory B and T cells to, for example, enable the production ofantibodies or other immune mediators specific for the HtrA polypeptideor fragment thereof.

As understood in the art, the term “vaccination” refers to the processof administering a vaccine to a person, whereas the term “immunization”refers to the stimulation of the immune system to produce an at leastpartially protective immune response following vaccination.

The terms “antigen” and “antigenic” are well understood in the art andrefer to the portion of a macromolecule which is specifically recognizedby a component of the immune system, e.g., an antibody or a T-cellantigen receptor. The term “antigen” refers to a peptide, a polypeptide,or other macromolecule to which an immune response can be induced in ahost. Thus the method of the invention may utilise an antigenic fragmentof an HtrA polypeptide. Preferably, the antigenic fragment is capable ofraising an immune response against a bacterial pathogen, for example abacterium from the genus Helicobacter including, but not limited to,Helicobacter pylori. In one embodiment, the antigen is an epitope of theHtrA polypeptide. In one embodiment, the antigenic fragment is 6 aminoacids in length, more preferably 7 amino acids in length, morepreferably 8 amino acids in length, more preferably 9 amino acids inlength, more preferably at least 10 amino acids in length. Alternativelythe antigenic fragment is at least 20, 30, 40, 50, 60, 70, 80, 90, 100or more amino acids in length. In an embodiment, the antigen whenadministered to a subject is able to elicit an immune response againstthe HtrA polypeptide.

The vaccines and/or immunogenic compositions of the invention may beadministered by any suitable delivery route, such as intradermal,mucosal e.g. intranasal, oral, intramuscular or subcutaneous. Otherdelivery routes are well known in the art. Vaccine preparation isgenerally described in Vaccine Design (“The subunit and adjuvantapproach” (eds Powell M. F. & Newman M. J.) (1995) Plenum Press, NewYork). In one embodiment, the immunogenic composition of the inventionis administered by the intramuscular delivery route. Intramuscularadministration may be to the thigh or the upper arm. Injection istypically via a needle (e.g. a hypodermic needle), but needle-freeinjection may alternatively be used. A typical intramuscular dose is 0.5ml.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO99/34850 and EP1092444, also the jetinjection devices described for example in WO 01/13977. Alternativemethods of intradermal administration of the vaccine preparations mayinclude conventional syringes and needles, or devices designed forballistic delivery of solid vaccines, or transdermal patches, or appliedto the surface of the skin (transdermal or transcutaneous delivery).

When the vaccines or immunogenic compositions of the present inventionare to be administered to the skin, or more specifically into thedermis, the vaccine is in a low liquid volume, particularly a volume ofbetween about 0.05 ml and 0.2 ml. Another suitable administration routeis the subcutaneous route. Any suitable device may be used forsubcutaneous delivery, for example classical needle. In one aspect ofthe invention, a needle-free jet injector service is used, for examplesuch as that published in WO 02/34317. In another aspect of theinvention, the device is pre-filled with the liquid vaccine formulation.

In one embodiment, the vaccine is administered intranasally. Typically,the vaccine is administered locally to the nasopharyngeal area.Preferred devices for intranasal administration of the vaccinesaccording to the invention are spray devices. Suitable commerciallyavailable nasal spray devices include Accuspray™ (Becton Dickinson).

The immunogenic composition and/or vaccine as described herein may be inthe form of a therapeutic or prophylactic composition or vaccine. Aprophylactic administration of an immunogenic composition or vaccinepreferably protects the recipient from the development of inflammationand/or a disease, or delays the development of inflammation and/ordisease, that would be caused by infection with bacteria comprising theHtrA polypeptide. A prophylactic composition or vaccine is administeredto a patient who is not known to be infected with the bacteria, or isknown to be free from infection with the bacteria, for example H.pylori, which expresses the HtrA polypeptide, or who has not yet beenexposed to the bacteria. Alternatively, a therapeutic immunogeniccomposition and/or vaccine is administered to a subject known orexpected to already be infected with the bacteria expressing the HtrApolypeptide, and who may already be exhibiting signs or symptoms ofinflammation and/or disease caused by infection with the bacteria. Thetherapeutic composition and/or vaccine prevents or delays theprogression of inflammation and/or disease caused by infection with thebacteria, or reduces the signs or symptoms of inflammation and/ordisease caused by infection with the bacteria expressing the HtrApolypeptide.

Adjuvants are useful for improving the immune response and/or increasingthe stability of vaccine preparations. Adjuvants are typically describedas non-specific stimulators of the immune system, but also can be usefulfor targeting specific arms of the immune system. One or more compoundswhich have this activity may be added to the vaccine. Therefore,particular vaccines of the present invention further comprise anadjuvant. Suitable vaccine adjuvants include, but are not limited to,aluminium salts (aluminium phosphate or aluminium hydroxide),monophosphoryl lipid A (for example 3D-MPL), saponins (for exampleQS21), oil in water emulsions, blebs or outer membrane vesiclepreparations from Gram negative bacterial strains (such as those taughtby WO02/09746), lipid A or derivatives thereof, alkyl glucosamidephosphates or combinations of two or more of these adjuvants. In oneembodiment the adjuvant is aluminium phosphate. In a further embodimentthe adjuvant comprises 100-750, 150-600, 200-500, 250-450, 300-400, oraround 350 μg aluminium as aluminium phosphate per human dose. In afurther embodiment the adjuvant is aluminium hydroxide.

An “ISCOM-like adjuvant” is an adjuvant comprising an immune stimulatingcomplex (ISCOM), which is comprised of a saponin, cholesterol, and aphospholipid, which together form a characteristic caged-like particle,having a unique spherical, caged-like structure that contributes to itsfunction. This term includes both ISCOM adjuvants, which are producedwith an antigen and comprise antigen within the ISCOM particle and ISCOMmatrix adjuvants, which are hollow ISCOM-type adjuvants that areproduced without antigen. In one embodiment, immunogenic compositionand/or vaccine as described herein comprises an ISCOM matrix particleadjuvant, such as ISCOMATRIX™, which is manufactured without antigen(ISCOM™ and ISCOMATRIX™ are the registered trademarks of CSL Limited,Parkville, Australia).

In certain embodiments, the adjuvant is a cytokine. Certain compositionsof the present invention comprise one or more cytokines, chemokines, orcompounds that induce the production of cytokines and chemokines, or apolynucleotide encoding one or more cytokines, chemokines, or compoundsthat induce the production of cytokines and chemokines. Examples ofcytokines include, but are not limited to granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), macrophage colony stimulating factor (M-CSF), colonystimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2),interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5),interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8),interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-11),interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 14 (IL-14),interleukin 15 (IL-15), interleukin 16 (IL-16), interleukin 17 (IL-17),interleukin 18 (IL-18), interferon alpha (IFNα), interferon beta (IFNβ),interferon gamma (IFNγ), interferon omega (IFNω), interferon tau (IFNτ),interferon gamma inducing factor I (IGIF), transforming growth factorbeta (TGF-β), RANTES (regulated upon activation, normal T-cell expressedand presumably secreted), macrophage inflammatory proteins (e.g., MIP-1alpha and MIP-1 beta), Leishmania elongation initiating factor (LEIF),and Flt-3 ligand.

In a further embodiment, the HtrA polypeptide or fragment thereof of atleast 10 amino acids may be conjugated to a bacterial toxoid, such as atoxoid from diphtheria, tetanus, E. coli (e.g., a heat labile toxin),cholera, H. pylori, or other pathogen. Furthermore, the HtrA polypeptideor fragment thereof of at least 10 amino acids may be conjugated to abacterial polysaccharide, such as the capsular polysaccharide fromNeisseria sp., Streptococcus pneumoniae or Haemophilus influenzae type-bbacteria.

An excipient is an inactive substance formulated alongside the activeingredient (“API”) of a medication, for the purpose of bulking-upformulations that contain potent active ingredients (thus often referredto as “bulking agents”, “fillers”, or “diluents”). Bulking up allowsconvenient and accurate dispensation of a drug substance when producinga dosage form. They also can serve various therapeutic-enhancingpurposes, such as facilitating drug absorption or solubility, or otherpharmacokinetic considerations. Excipients can also be useful in themanufacturing process, to aid in the handling of the active substanceconcerned such as by facilitating powder flowability or non-stickproperties, in addition to aiding in vitro stability such as preventionof denaturation over the expected shelf life. As known in the art, theselection of appropriate excipients also depends upon the route ofadministration and the dosage form, as well as the active ingredient andother factors.

Other suitable adjuvants, which sometimes have been referred to asimmune stimulants, include, but are not limited to: cytokines, growthfactors, chemokines, supernatants from cell cultures of lymphocytes,monocytes, cells from lymphoid organs, cell preparations and/or extractsfrom plants, bacteria or parasites (Staphylococcus aureus orlipopolysaccharide preparations) or mitogens.

A further aspect of the invention is a method of making the vaccineand/or immunogenic composition of the invention comprising the steps ofmixing an HtrA polypeptide or fragment thereof with a pharmaceuticallyacceptable excipient and/or adjuvant.

The HtrA polypeptide or fragment thereof of at least 10 amino acids canalso be administered via liposome carriers, which serve to target thepolypeptides to a particular tissue, such as lymphoid tissue, or totarget selectively to infected cells, as well as to increase thehalf-life of the polypeptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepolypeptide to be delivered is incorporated as part of a liposome, aloneor in conjunction with a molecule which binds to a receptor prevalentamong lymphoid cells (such as monoclonal antibodies which bind to theCD45 antigen or other costimulatory factor) or with other therapeutic orimmunogenic compositions. Thus, liposomes either filled or decoratedwith a desired polypeptide can be directed to the site of lymphoidcells, where the liposomes then deliver the polypeptide compositions.Liposomes for use in accordance with the invention are formed fromstandard vesicle-forming lipids, which generally include neutral andnegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally guided by consideration of, e.g.,liposome size, acid lability and stability of the liposomes in the bloodstream. A variety of methods are available in the art for preparingliposomes. A liposome suspension containing an HtrA polypeptide orfragment thereof of at least 10 amino acids may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the polypeptide beingdelivered, and the stage of the disease being treated.

In one embodiment, the immunogenic composition and/or vaccine asdescribed herein is administered together with, or in conjunction with,or as a combination with, a conventional therapy for treatment ofbacterial infection. For example, the conventional therapy may be anorganism-specific antibiotic. In the case of infection with H. pylori,the conventional therapy may be a combination of an antibiotic and anacid suppressor and/or stomach protector. Drugs that have been used totreat H. pylori infection include amoxicillin, clarithromycin,Rifabutin, Bismuth subsalicylate Furazolidone, Lactoferrin, Nitazoxanideand Levofloxacin. A combination that has been used to treat H. pyloriinfection includes Omeprazole (200 mg bd), Amoxycillin (1000 mg bd) andClarithromycin (500 mg bd).

Attenuated Bacteria

In one embodiment, an HtrA polypeptide or fragment thereof of at least10 amino acids is expressed by an attenuated bacterial (e.g., Salmonellasp.) or viral vaccine carrier (e.g., MVA). Methods of attenuating thevirulence of bacterial pathogens are known in the art. Typically,mutations are introduced into a bacterial genome to prevent or reduceexpression of toxins or other virulence genes to delete or inactivatethe gene. In some instances, the function of the gene is knocked-outcompletely. This may be achieved by abolishing synthesis of anypolypeptide at all from the gene or by making a mutation that results insynthesis of non-functional polypeptide. In order to abolish synthesisof polypeptide, either the entire gene or a portion, for example the5′-end, may be deleted. A deletion or insertion within the codingsequence of a gene may be used to create a gene that synthesizes onlynon-functional polypeptide (e.g., polypeptide that contains only theN-terminal sequence of the wild-type protein). In the case of a toxingene, the mutation may render the gene product non-toxic.

The present disclosure thus includes compositions comprising anattenuated bacterial or viral vaccine carrier which expresses an HtrApolypeptide or fragment thereof of at least 10 amino acids. The nucleicacid encoding the HtrA polypeptide may be in a vector or may beincorporated into the genome of the host carrier, for instance, byhomologous recombination or genome editing (genome editing withengineered nucleases (GEEN). Examples of engineered nucleases suitablefor genome editing include Transcription Activator-Like EffectorNucleases (TALENs), and the CRISPR/Cas system. In one embodiment of theinvention, the vaccine composition comprises an attenuated Salmonellavaccine carrier comprising an attenuating mutation in a SalmonellaPathogenicity Island 2 region and, optionally, one or more additionalattenuating mutations (for instance, a mutation in one or more of thearoA, aroC or sod genes) and a nucleic acid encoding an HtrA polypeptideor a fragment thereof of at least 10 amino acids. The attenuated livevaccine compositions of the present invention can further comprise, forinstance, a pharmaceutically acceptable carrier, diluent and/oradjuvant.

Antibody Therapy

In addition to prophylactic and therapeutic vaccination, the presentinvention also includes antibody therapy for treatment of a patienthaving or suspected of having bacteria induced inflammation and/ordisease. As used herein, the term “antibody therapy” includes referenceto a treatment protocol or treatment regimen that includes theadministration of an antibody to a subject. As would be understood inthe art, the term “antibody therapy” encompasses both passiveimmunotherapy and passive immunoprophylaxis.

The term “antibody” as used herein refers to an immunoglobulin,immunoglobulin fragment, or immunoglobulin-like molecule capable ofbinding to a target, for example a bacterial HtrA polypeptide orfragment thereof. Thus, the term “antibody” encompasses not only intactpolyclonal or monoclonal antibodies, but also bispecific antibodies,diabodies, triabodies, heteroconjugate antibodies, chimeric antibodies,humanized and human antibodies including intact molecules as well asfragments thereof, such as Fab, Fab′, F(ab′)₂, Fv, and scFv which arecapable of binding the HtrA polypeptide or fragment thereof.

As used herein, the term “monoclonal antibody” (mAb) refers to anantibody that is derived from a single copy or clone including, forexample, any eukaryotic, prokaryotic, or phage clone, and not the methodby which it is produced. mAbs used in the present invention preferablyexist in a homogeneous or substantially homogeneous population. CompletemAbs contain two heavy chains and two light chains. Thus, fragments ofsuch monoclonal antibodies include, for example, Fab fragments, Fab′fragments, F(ab′)₂, single chain Fv fragments, and one-armed antibodiescomprising a light chain and a heavy chain. Monoclonal antibodies andantigen-binding fragments thereof can be produced, for example, byrecombinant technologies, phage display technologies, synthetictechnologies, e.g., CDR-grafting, or combinations of such technologies,or other technologies known in the art.

Antibodies exhibiting desired functional properties, i.e., binding to anHtrA polypeptide or fragment thereof, can be generated by conventionalmethods. For example, mice can be immunized with an HtrA polypeptide orfragments thereof, the resulting antibodies can be recovered andpurified, and determination of whether they possess the desired bindingand functional properties can be assessed using known methods.Antigen-binding fragments can also be prepared by conventional methods.Methods for producing and purifying antibodies and antigen-bindingfragments are well known in the art and can be found, for example, inHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 5-8 and 15,ISBN 0-87969-314-2.

The phrase “humanized antibodies” refers to monoclonal antibodies andantigen binding fragments thereof that have framework regions that aresubstantially human or fully human surrounding CDRs derived from anon-human antibody. “Framework region” or “framework sequence” refers toany one of framework regions 1 to 4. Humanized antibodies and antigenbinding fragments include molecules wherein any one or more of frameworkregions 1 to 4 is substantially or fully human, i.e., wherein any of thepossible combinations of individual substantially or fully humanframework regions 1 to 4, is present. For example, this includesmolecules in which framework region 1 and framework region 2, frameworkregion 1 and framework region 3, framework region 1, 2, and 3, etc., aresubstantially or fully human. Substantially human frameworks are thosethat have at least about 80% sequence identity to a known human germlineframework sequence. Preferably, the substantially human frameworks haveat least about 85%, at least about 90%, at least about 95%, or at leastabout 99% sequence identity to a known human germline frameworksequence.

Fully human frameworks are those that are identical to a known humangermline framework sequence. Human framework germline sequences can beobtained from ImMunoGeneTics (IMGT) via their website or from TheImmunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc,Academic Press, 2001, ISBN 012441351. For example, germline light chainframeworks can be selected from the group consisting of: A11, A17, A18,A19, A20, A27, A30, LI, L1I, L12, L2, L5, L15, L6, L8, O12, O2, and O8,and germline heavy chain framework regions can be selected from thegroup consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VHI-46,VH3-9, VH3-66, VH3-74, VH4-31, VHI-18, VHI-69, VI-13-7, VH3-11, VH3-15,VH3-21, VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, and VH5-5I.

Humanized antibodies can be generated using several different methods.In one approach, the parent antibody compound CDRs are grafted into ahuman framework that has a high sequence identity with the parentantibody compound framework. The sequence identity of the new frameworkwill generally be at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identical to the sequenceof the corresponding framework in the parent antibody compound. In thecase of frameworks having fewer than 100 amino acid residues, one, two,or three amino acid residues can be changed. This grafting may result ina reduction in binding affinity compared to that of the parent antibody.If this is the case, the framework can be back-mutated to the parentframework at certain positions based on specific criteria disclosed byQueen et al. (1991). Additional references describing methods useful inhumanizing mouse antibodies include U.S. Pat. Nos. 4,816,397; 5,225,539;and 5,693,761; computer programs ABMOD and ENCAD as described in Levitt(1983); and the method of Winter and co-workers (Jones et al., 1986).

An antibody or antibody fragment, or immunogenic or pharmaceuticalcomposition comprising the same, may be administered by parenteralroutes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular,or transdermal). An antibody, or antigen-binding fragment thereof, ofthe present invention may be administered to a patient alone or incombination with a pharmaceutically acceptable carrier and/or excipientin single or multiple doses. Pharmaceutical compositions of the presentinvention can be prepared by methods well known in the art (e.g.,Remington: The Science and Practice of Pharmacy, 19^(th) ed. (1995), A.Gennaro et al., Mack Publishing Co.) and comprise an antibody, asdisclosed herein, and one or more pharmaceutically acceptable carriers,diluents, or excipients.

In one embodiment there is provided a composition, e.g., apharmaceutical composition, containing one or a combination ofantibodies that bind an HtrA polypeptide, fragment or epitope thereof.The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19^(th)Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

Pharmaceutical compositions also can be administered in combinationtherapy, i.e., combined with other agents. For example, the combinationtherapy can include a composition with at least one anti-HtrA antibodyand an anti-inflammatory agent. In one embodiment such anti-inflammatoryagents include a steroidal drug or a NSAID (nonsteroidalanti-inflammatory drug). Examples of agents include, aspirin and othersalicylates, Cox-2 inhibitors, such as rofecoxib (Vioxx) and celecoxib(Celebrex), NSAIDs such as ibuprofen (Motrin, Advil), fenoprofen(Nalfon), naproxen (Naprosyn), sulindac (Clinoril), diclofenac(Voltaren), piroxicam (Feldene), ketoprofen (Orudis), diflunisal(Dolobid), nabumetone (Relafen), etodolac (Lodine), oxaprozin (Daypro),and indomethacin (Indocin).

In one embodiment, an antibody that binds HtrA polypeptide isadministered together with, or in conjunction with, or as a combinationwith, a conventional therapy for treatment of bacterial infection.

DNA Vaccines

DNA vaccination involves the direct in vivo introduction of DNA encodingan antigen into cells and/or tissues of a subject for expression of theantigen by the cells of the subject's tissue. Such vaccines are termedherein “DNA vaccines” or “nucleic acid-based vaccines.” Examples of DNAvaccines are described in U.S. Pat. Nos. 5,939,400, 6,110,898, WO95/20660 and WO 93/19183. The ability of directly injected DNA thatencodes an antigen to elicit a protective immune response has beendemonstrated in numerous experimental systems.

A factor known to affect the immune response elicited by DNAimmunization is the method of DNA delivery, for example, parenteralroutes can yield low rates of gene transfer and produce considerablevariability of gene expression. High-velocity inoculation of plasmids,using a gene-gun, enhanced the immune responses of mice, presumablybecause of a greater efficiency of DNA transfection and more effectiveantigen presentation by dendritic cells. Vectors containing the nucleicacid-based vaccine of the invention may also be introduced into thedesired host by other methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, lipofection (lysosome fusion),or a DNA vector transporter.

Treatment of Disease

The present inventors have determined that an HtrA polypeptide orfragment thereof of at least 10 amino acids, polynucleotide encoding anHtrA polypeptide or fragment thereof of at least 10 amino acids, animmunogenic composition, pharmaceutical composition, and/or vaccine asdescribed herein can be administered to a subject, preferably a humansubject, in order to treat or prevent bacteria induced inflammationand/or diseases resulting from infection with a bacterium that expressesthe HtrA polypeptide. The present inventors have found that thistreatment reduces or delays the development of signs or symptoms ofinflammation and/or disease caused by infection with a bacteriumexpressing an HtrA polypeptide, despite the treatment not eradicating orpreventing infection with the bacteria.

As would be understood in the art, the term “bacteria inducedinflammation” refers to a biological response of tissues to harmfulstimuli from bacteria, and includes both acute and chronic inflammation.The process of acute inflammation may be initiated by activation ofinnate immune cells already present in all tissues, mainly residentmacrophages, dendritic cells, histiocytes, Kupffer cells and mastocytes.These cells present on their surfaces certain receptors named patternrecognition receptors (PRRs), which recognize molecules that are broadlyshared by pathogens but distinguishable from host molecules,collectively referred to as pathogen-associated molecular patterns(PAMPs). At the onset of an infection may cells may undergo activation(one of their PRRs recognize a PAMP) and release inflammatory mediatorsresponsible for the clinical signs of inflammation. Activation of thisinnate immune response then supports the activation of an acquiredimmune response, which particularly involves T-helper (Th) lymphocytes.H. pylori gastritis involves a mixed Th1 and Th17 type immune responsemarked by the pro-inflammatory cytokines IFNγ and IL-17, respectively.The severity of gastritis caused by these responses is regulated byproduction of immunosuppressive IL-10. Th2-type responses, for example,IL-13, also inhibit such responses. Vasodilation and its resultingincreased blood flow causes the redness (rubor) and increased heat(calor). Increased permeability of the blood vessels results in anexudation (leakage) of plasma proteins and fluid into the tissue(edema), which manifests itself as swelling (tumor). Some of thereleased mediators such as bradykinin increase the sensitivity to pain(hyperalgesia, dolor). The mediator molecules also alter the bloodvessels to permit the migration of leukocytes, mainly neutrophils,outside of the blood vessels (extravasation) into the tissue. Theneutrophils migrate along a chemotactic gradient created by the localcells to reach the site of injury.

In addition to cell-derived mediators, several acellular biochemicalcascade systems consisting of preformed plasma proteins act in parallelto initiate and propagate the inflammatory response. These include thecomplement system activated by bacteria. The exudative componentinvolves the movement of plasma fluid, containing important proteinssuch as fibrin and immunoglobulins (antibodies), into inflamed tissue.This movement is achieved via the chemically induced dilation andincreased permeability of blood vessels, which results in a net loss ofblood plasma. The increased collection of fluid into the tissue causesit to swell (edema). This extravasated fluid is funneled by lymphaticsto the regional lymph nodes, flushing cells exposed to H. pylori antigenalong to start the recognition and attack phase of the adaptive immunesystem.

Acute inflammation is characterized by marked vascular changes,including vasodilation, increased permeability and increased blood flow,which are induced by the actions of various inflammatory mediators.Vasodilation occurs first at the arteriole level, progressing to thecapillary level, and brings about a net increase in the amount of bloodpresent, causing the redness and heat of inflammation. Increasedpermeability of the vessels results in the movement of plasma into thetissues, with resultant stasis due to the increase in the concentrationof the cells within blood—a condition characterized by enlarged vesselspacked with cells. Stasis allows leukocytes to marginate (move) alongthe endothelium, a process critical to their recruitment into thetissues.

Specific patterns of acute and chronic inflammation are seen duringparticular situations that arise in the body, such as when inflammationoccurs on an epithelial surface, or pyogenic bacteria are involved.

1) Granulomatous inflammation: Characterized by the formation ofgranulomas, they are the result of a limited but diverse number ofdiseases, which include among others tuberculosis, leprosy, sarcoidosis,and syphilis.

2) Fibrinous inflammation: Inflammation resulting in a large increase invascular permeability allows fibrin to pass through the blood vessels.If an appropriate procoagulative stimulus is present, such as cancercells, a fibrinous exudate is deposited. This is commonly seen in serouscavities, where the conversion of fibrinous exudate into a scar canoccur between serous membranes, limiting their function. The depositsometimes forms a pseudomembrane sheet. During inflammation of theintestine (Pseudomembranous colitis), pseudomembranous tubes can beformed.

3) Purulent inflammation: Inflammation resulting in large amount of pus,which consists of neutrophils, dead cells, and fluid. Infection bypyogenic bacteria such as staphylococci is characteristic of this kindof inflammation. Large, localised collections of pus enclosed bysurrounding tissues are called abscesses.

4) Serous inflammation: Characterized by the copious effusion ofnon-viscous serous fluid, commonly produced by mesothelial cells ofserous membranes, but may be derived from blood plasma. Skin blistersexemplify this pattern of inflammation.

5) Ulcerative inflammation: Inflammation occurring near an epitheliumcan result in the necrotic loss of tissue from the surface, exposinglower layers. The subsequent excavation in the epithelium is known as anulcer.

In one embodiment, the “bacteria induced” inflammation is gastritis(i.e., inflammation of the lining of the stomach) or duodenitis (i.e.,inflammation of the duodenum). Erosive gastritis is gastric mucosalerosion caused by damage to mucosal defenses. Chronic gastritis refersto a wide range of problems of the gastric tissues.

In the case of gastritis induced by infection or colonization withHelicobacter pylori, acute infection may appear as an acute gastritiswith abdominal pain (stomach ache) or nausea. Where this develops intochronic gastritis, the symptoms, if present, are often those ofnon-ulcer dyspepsia: stomach pains, nausea, bloating, belching, andsometimes vomiting or black stool. Individuals infected with H. pylorihave a 10 to 20% lifetime risk of developing peptic ulcers and a 1 to 2%risk of acquiring stomach cancer. Inflammation of the pyloric antrum ismore likely to lead to duodenal ulcers, while inflammation of the corpus(body of the stomach) is more likely to lead to gastric ulcers andgastric adenocarcinoma.

Mucous gland metaplasia, the reversible replacement of differentiatedcells, occurs in the setting of severe damage of the gastric glands,which then waste away (atrophic gastritis) and are progressivelyreplaced by mucous glands. Gastric ulcers may develop. Thus, in oneembodiment, the bacteria induced inflammation is atrophic gastritis.

In one embodiment, there is provided a method of treating or preventinga disease caused by Helicobacter pylori infection in a subject. As knownin the art, diseases caused by infection with H. pylori includeinflammation, gastritis, duodenitis, atrophic gastritis, gastric ulcer,duodenal ulcer, gastric cancer, and/or mucosa-associated-lymphoid-tissue(MALT) lymphoma.

EXAMPLES Example 1. Preparation of HtrA Antigen and Vaccinations

Recombinant HtrA (including mutant) was produced as described by Loweret al. (2008). The gene hp1018 was amplified from the genomic DNA of H.pylori strain 26695 by standard PCR using the Pfx DNApolymerase(Invitrogen, Karlsruhe, Germany). The following primers were used:hp1018 for: 5′-GGC TAT GGA TAA GGA TCA ACG C-3′ (SEQ ID NO:37),hp1018rev: 5′-CCA CCG CCT TAA TAG AGT CCT T-3′ (SEQ ID NO:38). The PCRproduct, having a calculated length of 333 bases, was submitted to acommercial provider (GENterprise, Mainz, Germany) for sequencing.

The construct Hp1018/19Dsp was amplified from genomic DNA of H. pyloristrain 26695 using the primers 5′-aaggatccggcaatatccaaatccagagcatg-3′(SEQ ID NO:39) and 5′-aagaattcgacccacccctatcatttcacc-3 (SEQ ID NO:40)with Pfx DNA polymerase in supplied buffer with 26 PCR Enhancer(Invitrogen). The amplified BamH1/EcoR1 flanked PCR product was thenligated into the pGEM-T Easy plasmid (Promega), subcloned into thepGEX-6P-1 plasmid DNA (GE Healthcare Life Sciences) and transformed inE. coli BL21. The construction of the protease-inactiveHp1018/19DspS205A protein, serine 205 was mutated to alanine using theQuikChangeH Lightning Site-Directed Mutagenesis Kit (Stratagene)according to the manufacturer's instructions. For heterologousoverexpression and purification of GST-Hp1018/19Dsp, transformed E. coliwas grown in 500 ml TB medium to an OD₅₅₀ of 0.6 and the expression wasinduced by the addition of 0.1 mM isopropylthiogalactosid (IPTG). Thebacterial culture was pelleted at 4000 g for 30 minutes and lysed in 25ml PBS by sonification. The lysate was cleared by centrifugation and thesupernatant was incubated with glutathione sepharose (GE Healthcare LifeSciences) at 4° C. overnight. The fusion protein was either eluted with10 mM reduced glutathione for 10 minutes at room temperature or cleavedwith 180 U Prescission Protease for 16 h at 4° C. (GE Healthcare LifeSciences). Elution and cleavage products were analyzed by SDS PAGE andzymography.

Specific-pathogen free age-matched female C57BL/6 mice, bred within theVeterinary Science animal house, University of Melbourne, Parkville,were vaccinated twice with recombinant HtrA antigen (SEQ ID NO:1), threeweeks apart via the nasal route. For nasal vaccination, the vaccine in a30 μL volume was slowly applied to the external nares of the consciousmouse. Mice were immunized subcutaneously with 100 μg of formalin fixedH. pylori, 100 μg of alum, or 10 μg of HtrA plus 100 μg of alum.

Example 2. H. pylori Cultivation and Challenge of Mice

H. pylori strain SS1 was cultivated in brain heart infusion broth (BHI;Oxoid, Basingstoke, UK) containing 5% horse serum (JRH Biosciences,USA), 0.02% amphotericin B and Skirrow's Selective Supplements, undermicroaerophilic conditions at 37° C.

H. pylori lysate (HpL) was prepared by adding 0.1 mm glass beads(Daintree Scientific, St. Helens, TAS, Australia) to the bacteria andpulsing at 60 m/s using the FastPrep FP120 (Thermo Savant, Waltham,Mass., USA) in 30-second cycles with incubation on ice between cycles,until bacteria were lysed. Sterility was confirmed by culturing on horseblood agar plates. Protein levels were quantified using the BCA ProteinAssay Kit (Pierce, Rockford, Ill., USA).

For formalin-fixation for vaccination, bacteria were suspended in 0.01 Mformaldehyde in PBS and adjusted to an OD₆₀₀ of 1.5. After 2 hoursgentle shaking at 37° C., the bacteria were shaken overnight at roomtemperature, washed 3 times in PBS and resuspended at 10⁸ bacteria/mL.

C57BL/6 mice used in these studies were age-matched females obtainedfrom the Walter and Eliza Hall Institute, Melbourne. All animalexperiments were approved by University of Melbourne Animal EthicsCommittee.

Mice were infected intragastrically once with 10⁷ H. pylori strain SS1suspended in 0.1 mL BHI. At completion of the experiment, mice wereeuthanized by CO₂ asphyxiation, and the stomachs opened along the innercurvature and divided into two halves. One half was used to quantifybacterial levels by colony-forming assay. The other half was fixed inneutral buffered formalin for histological assessment of gastritis.

Example 3. Colony Forming Assay

Four weeks after challenge, stomachs were opened along the innercurvature and divided into two halves. One half was placed in BHI brothand homogenized (GmbH Polytron® homogeniser, Kinematica, Switzerland).Ten-fold serial dilutions were prepared in BHI broth and aliquots spreadover Glaxo selective supplement agar plates (Blood Agar Base No. 2, with5% horse blood, 3.75 μg/mL Amphostat B, 12 μg/mL vancomycin, 0.4 μg/mLpolymyxin B, 20 μg/mL bacitracin and 1.3 μg/mL nalidixic acid). After 5days culture, colonies were counted and the number of colony formingunits per stomach calculated.

Example 4. Gastric Pathology

Gastritis was assessed histologically as described previously (Sutton etal., 2001). The second halves of each stomach were fixed in 10% neutralbuffered formalin, embedded in paraffin and 4 μm-thick sections cut. Forassessment of gastritis, sections were stained with H&E and scoredblinded under light microscopy. Inflammation was assessed in 2 separatetissue sections for each animal using 3 parameters. (1) cellularinfiltration (migration of lymphocytes and neutrophils into the laminapropria) graded from 0 to 6, where 0, none; 1, mild multifocal; 2, mildwidespread; 3, mild widespread and moderate multifocal; 4, moderatewidespread; 5, moderate widespread and severe multifocal; and 6, severewidespread; (2) “mucus metaplasia” (large, pale, globular cells in thecorpus) and (3) “atrophy” (loss of parietal and chief cells). Metaplasiaand atrophy were graded from 0 to 3 (0, absent; 1, mild; 2, moderate; 3,severe).

Example 5. Gastric Cytokines

Cell debris was removed from stomach homogenates by centrifugation andprotein concentrations in the supernatant quantified using a BCA proteinassay kit (Pierce, Rockford, Ill., USA) prior to ELISA. Cytokineconcentrations were determined by coating 96-well Maxisorp plates (Nunc,Roskilde, Denmark) with purified anti-mouse IL-13 (50 ng/well;eBioscience, San Diego, Calif., USA), IL-10 (100 ng/well; BDBiosciences, San Jose, Calif., USA), IFNγ (100 ng/well; BD Biosciences)or IL-17A (50 ng/well; eBioscience) overnight in bicarbonate coatingbuffer, pH 9.6. Plates were blocked with 1% BSA (Sigma) in PBS (blocker)for one hour prior to addition of samples in duplicate for three hoursat room temperature or 4° C. overnight. Captured cytokines were thenlabelled with biotinylated anti-mouse IL-13 (25 ng/well; eBioscience),IL-10 (50 ng/well; BD Bio-sciences), IFNγ (50 ng/well; BD Biosciences)or IL-17A (25 ng/well; eBioscience) in blocker for one hour prior to theaddition of 50 μL horseradish peroxidase conjugated streptavidin(Pierce) 1/5000 in blocker for 30 min. Colour was developed with 100 μLof TMB solution prepared as 0.1% of 10 mg/mL TMB (Sigma) in DMSO and0.006% hydrogen peroxide in phosphate-citrate buffer, pH 5.0, and thereaction stopped with an equal volume of 2 M sulphuric acid prior toreading absorbance at 450 nm. Sample concentration was determinedagainst a standard curve of recombinant IL-10, IFNγ (BD Biosciences),IL-13 and IL-17A (eBioscience).

Example 6. HtrA Serum Inhibition Assay

The EnzChek® peptidase/protease assay kit (E33758) was used to measurethe ability of sera from the above vaccinated mice to block proteolyticactivity of HtrA. Sera from vaccinated/infected mice (collected fourweeks after challenge) were diluted 1:5 and mixed with either HtrA orPBS (control) using component B (digestion buffer) of the peptidase kitfor 30 mins. The proteolytic activity of HtrA was then assessed bymeasuring cleavage of substrate following incubation for 60 min (as perkit recommendations). Absorbance was read at 502/528 nm using a TECANInfinite M200Pro reader and data analysed using Magellan 7.1 SPIsoftware.

Example 7. Quantification of Antibody Response to Infection

Sera were collected by cardiac puncture. Intestinal mucus scrapings werecollected from the lower 10 cm of the longitudinally opened smallintestine using a scalpel blade, weighed, then mixed with an equalvolume of PBS containing complete mini-EDTA-free proteinase inhibitorcocktail (Roche Diagnostics, Mannheim, Germany). Anti-Helicobacterantibody levels were determined by direct ELISA. Maxisorp immunoplates(Nunc, Denmark) were coated overnight with 50 μg/well of H. pylorilysate in bicarbonate buffer pH9.6. Plates were blocked for 1 hour atRT. Samples were serially diluted 1/10 in blocker and 50 added toduplicate wells, before incubation at RT for 1 hour. Captured antibodieswere labelled with horseradish peroxidase conjugated goat-anti-mouse IgG(4 ng/well; Pierce), goat-anti-mouse IgG1 (8 ng/well, Southern Biotech,Birmingham, Ala., USA) or IgA (10 ng/well, Southern Biotech) in blockerfor 1 hour at RT. Colour was developed and read as above and end pointtitres calculated.

Example 8. Western Blotting

H. pylori lysate (20 μg/lane) was separated on 8% SDS-PAGE gels thentransferred to nitrocellulose membranes (Amersham Biosciences).Membranes were blocked with 5% skim milk and probed with intestinalscrapings diluted 1/1,000. Bound primary antibodies were detected withhorseradish peroxidase-conjugated anti-mouse antibodies (Dako). Labelledproteins were visualised by incubating the membrane in ECL Prime reagent(Amersham Biosciences) and using an ImageQuant LAS 4000 (GE Healthcare).All gels were exposed for 10 seconds.

Example 9. Statistics

Statistical analyses were performed using the Statistical Package forthe Social Sciences software, version 21.0.

Example 10. Results

Prophylactic Vaccination with HtrA Induces Minimal Protection

Groups of mice were vaccinated twice via the nasal route with killed H.pylori plus cholera toxin (CT) adjuvant (positive control) or HtrA plusCT. Negative controls were sham dosed with PBS. Four weeks after thelast dose, all mice were challenged with live H. pylori strain SS1.Bacterial burdens in mouse stomachs were assessed four weeks later bycolony forming assay. Vaccinations reduced bacterial colonisationcompared to the negative control group (FIG. 1; **p<0.01, ***p<0.0001;ANOVA).

Post Immunisation Gastritis

Stomachs from the mice referred to in FIG. 1 were assessedhistologically for gastritis. As expected, mice vaccinated with killedH. pylori plus CT before challenge had a significantly increasedseverity of gastritis (increased atrophy and metaplasia, key gastritisreadouts) compared to the negative control group (*Mann-Whitney; FIG.2). However the gastritis in mice vaccinated with HtrA plus CT wasindistinguishable from the negative control group. In other words,despite the protective immunity shown in FIG. 1, these mice did notdevelop post-immunisation gastritis.

This was unlikely to be related to the slightly lower level ofprotection observed in the HtrA plus CT group compared to the positivecontrol group; the present inventor has performed countless similarexperiments with recombinant antigens testing H. pylori vaccines and notseen this previously. It is well recognised in the literature that H.pylori colonisation does not correlate with gastritis severity.

Suppression of Gastric Cytokine Levels Post-Immunisation

H. pylori infection induces a mixed Th1 and Th17 response in the gastrictissues of both mice and humans. IL-10 is known to be an importantsuppressor of H. pylori-induced gastritis. IL-13 is the main marker usedfor measuring Th2-type responses. To explore this reduced gastritisfurther, cytokines in the gastric homogenates of the above mice werequantified by ELISA.

As expected, gastric tissues from the infection control (PBS) andpositive control vaccine (H. pylori+CT) groups presented with highlevels of Th1 (IFNγ) and Th17 (IL-17A) cytokines. This particularexperiment did not contain an uninfected control group, but years ofexperiments indicate these cytokines are not normally easily detected ingastric homogenates. IL-10 and IL-13 were also elevated; this is alsoexpected, as these cytokines increase to suppress the inflammatorysignals induced by Th1 and Th17-type cytokines.

The completely unexpected observation was that vaccination with HtrA+CTsignificantly suppressed levels of these cytokines in infected mice toextremely low or undetectable levels (**p<0.01, ***p<0.0001; ANOVA)(FIG. 3). This is the first time a vaccine has been demonstrated tosuppress H. pylori-induced gastritis.

Recombinant HtrA (rHtrA) Vaccination Induces a Neutralising AntibodyResponse

The present inventor hypothesized that the vaccine was suppressinggastritis and gastric cytokines levels by inducing antibodies thatneutralised HtrA activity. To test this, an in vitro assay for HtrAactivity was developed to assess the inhibitory ability of sera from theabove mice. The results in FIG. 4 show that sera from mice vaccinatedwith HtrA then challenged with H. pylori did indeed inhibit the abilityof HtrA protease to cleave a target substrate (***ANOVA). This was incontrast to sera from the other groups.

Recombinant HtrA (rHtrA) Vaccination Induces Antibodies Against HtrA

A group of mice was vaccinated three times with rHtrA+CT via the nasalroute. Negative controls were sham dosed with PBS. Sera from thevaccinated mice contained antibodies that specifically detected HtrA(FIG. 5).

Therapeutic HtrA Vaccination by Injection Route

Groups of mice were infected with H. pylori before being subcutaneouslyinjected with either alum adjuvant alone (negative control), rHtrA andalum or rHtra^(sa) and alum (rHtra^(sa) is identical to rHtrA except fora single amino acid substitution of serine to alanine which makes theenzyme inactive). Four weeks after the last vaccination, stomachs wereremoved and bacterial colonization quantified by colony-forming assay.There was no difference in H. pylori colonization levels between any ofthe groups (FIG. 6).

Therapeutic HtrA Vaccination by the Injection Route does Protect AgainstH. pylori-Induced Gastritis

The severity of gastritis in the stomachs of therapeutically vaccinatedmice described in FIG. 6 was quantified histologically. Despite havingno effect on colonization (FIG. 6), vaccination with either active rHtraor inactive rHtrA^(sa) resulted in protection against the development ofH. pylori-induced atrophic gastritis. (FIGS. 7A-7B).

Therapeutic HtrA Vaccination by the Injection Route Protects Against H.pylori-Induced Gastritis without a Significant Increase in GastricCytokines

Cytokine levels in the gastric homogenates from the mice described inFIG. 6 were quantified by ELISA. Vaccination with either rHtrA groupproduced no significant increase in key gastric cytokines, particularlycompared with the main negative control group vaccinated with alum alone(FIG. 8).

Therapeutic rHtrA Vaccination by the Injection Route Induces Antibodiesin the Gastrointestinal Tract Specific for Native HtrA

Intestinal scrapings from the mice described in FIG. 6 were tested forthe presence of antibodies against HtrA. Western blot analysis ofintestinal scrapings against H. pylori lysate showed that only infectedmice therapeutically vaccinate with rHtrA had detectable antibodiesagainst HtrA (FIG. 9). Importantly this also demonstrates thatvaccination with recombinant HtrA induced mucosal antibodies againstnative H. pylori HtrA.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the scope of theinvention as broadly described. The present embodiments are, therefore,to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

The present application claims priority from AU 2014902493 filed 30 Jun.2014, the entire contents of which are incorporated herein by reference.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

1. A method of treating or preventing Helicobacter pylori-inducedinflammation in a subject, the method comprising administering to thesubject a bacterial HtrA polypeptide, wherein the bacterial HtrApolypeptide comprises an amino acid sequence at least 90% identical toany one of SEQ ID NOs: 1-6.
 2. The method of claim 1, wherein the methodcomprises administering a pharmaceutical composition comprising thebacterial HtrA polypeptide and a pharmaceutically acceptable excipientand/or adjuvant.
 3. (canceled)
 4. The method of claim 1, wherein thebacterial HtrA polypeptide comprises an amino acid sequence at least 95%identical to any one of SEQ ID NOs:1-6.
 5. The method of claim 1,wherein the Helicobacter pylori-induced inflammation is gastritis and/orduodenitis.
 6. The method of claim 5, wherein the gastritis is atrophicgastritis. 7-25. (canceled)
 26. A method of treating or preventing adisease caused by Helicobacter pylori infection in a subject, the methodcomprising administering to the subject a bacterial HtrA polypeptide,wherein the bacterial HtrA polypeptide comprises an amino acid sequenceat least 90% identical to any one of SEQ ID NOs: 1-6. 26A. The method ofclaim 26, wherein the method comprises administering to the subject apharmaceutical composition comprising the bacterial HtrA polypeptide.27. The method of claim 26, wherein the bacterial HtrA polypeptidecomprises an amino sequence at least 95% identical to any one of SEQ IDNOs:1-6.
 28. The method of claim 26A, wherein the pharmaceuticalcomposition comprises a pharmaceutically acceptable excipient and/oradjuvant
 29. The method of claim 26, wherein the disease caused byHelicobacter pylori infection is inflammation, gastritis, gastric ulcer,duodenal ulcer, gastric cancer, and/or mucosa-associated-lymphoid-tissue(MALT) lymphoma.
 30. The method of claim 1, wherein the bacterial HtrApolypeptide is an enteropathogenic bacterial HtrA polypeptide.
 31. Themethod of claim 30, wherein the enteropathogenic bacterial HtrApolypeptide is selected from the group consisting of Helicobacter pyloriHtrA, Escherichia coli HtrA, Shigella flexneri HtrA, and Campylobacterjejuni HtrA.
 32. The method of claim 26, wherein the bacterial HtrApolypeptide is an enteropathogenic bacterial HtrA polypeptide.
 33. Themethod of claim 32, wherein the enteropathogenic bacterial HtrApolypeptide is selected from the group consisting of Helicobacter pyloriHtrA, Escherichia coli HtrA, Shigella flexneri HtrA, and Campylobacterjejuni HtrA.